bacillus anthracis - gmch lectures/microbiology/27 bacillus... · historical importance first...
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Bacillus Anthracis
HISTORICAL IMPORTANCE
First pathogenic bacteria to be seen under
microscope (Pollender 1849)
• First demonstration of blood borne transmission
(Davaine 1850)
• The first bacterium shown to be the cause of a
disease- Koch’s Postulate
• In 1877, Robert Koch grew the organism in pure
culture, demonstrated its ability to form endospores
• First effective vaccine (Pasteur 1881)
Robert Koch
Louis Pasteur
AGENT Bacillus anthracis Gram positive rods 3-10 micron by 1-1.6 micron
Aerobic bacilli forming heat-resistant spores
Non acid fast, Non-motile
Capsule (poly d-glutamate-protein) (10-25% CO2 bicarbonate, serum albumin
charcoal, starch
Spores
Aerobic bacilli forming heat-resistant spores Formed in culture/soil survive in soil for decades Never formed in animal body Occurs under un-favourable conditions Encouraged by DW, 2% NaCl, oxalated agar Inhibited by calcium chloride central or subterminal, oval Of same width as the bacillary body-no bulging resistant to drying/UV/gamma rays/heat
Bacillus anthracis. Gram stain. The cells have characteristic squared ends. The endospores are ellipsoidal shaped and located centrally in the sporangium. The spores are highly refractile to light and resistant to staining.
AGENT (contd…) Cultural characteristics 35-37˚ C Optimum for sporulation- 25-30˚C Good growth occurs on ordinary
media grey white (sheep blood agar) Irregular, non-haemolytic Tenacious, rough
LPF : Medusa head colony Edge of a colony is composed of long,
interlacing chains of bacilli resembling locks of matted hair
PLET medium
SPORES SURVIVE FOR MANY YEARS ( DRY STATE AND SOIL )
• DUCKERING: 2% Formaldehyde kills spores at 30-40˚C for 20 mins for disinfection of wool and as 0.25% at 60˚C for 6 hrs for animal hair and bristles
• 4% KMnO4 kills spores in 15 mins
PATHOGENICITY
HOST
In nature, primarily an infection of : herbivores :cattle, horse, goat, sheep, - from soil they graze, wild animals-carnivores
Farmers
Animal product handlers :meat, hide, wool, hair, bones etc.
Healthcare workers
Lab personel
? Common man : threat of bioterrorism
ENVIRONMENT Favorable factors for spore formation & survival
- abundant rainfall following a period of drought- soil pH >6 , abundant organic matter- improper disposal of animal carcasses (ideally incineration or rendering needs to be done)
• Human-animal interaction
• Asymmetric warfare
VIRULENCE FACTORS
Anthrax Toxin – Complex of proteins ( all the components thermolabile)• Edema factor (Factor I)• Protective factor (Factor II)• Lethal Factor (Factor III)
Protein capsule – Poly D Glutamic acid capsule- Inhibits phagocytosis ( Unencapsulated strains –
nonpathogenic)
Anthrax Toxin
Protective antigen : Binds plasma membrane of target cells
Cleaved to 2 fragments ( cellular trypsin or proteases)
Larger fragment is attached to cell surface – binding domain for LF & EF
Specific receptor mediated endocytosis of LF & EF
EDEMA FACTOR( Edema Factor + Protective Ag = Edema toxin)
Calmodulin dependent adenyl cyclase
Increased cellular cAMP Edema Impaired Neutrophil function
Depletes ATP from Macrophages
LETHAL FACTOR( Lethal Factor + Protective Ag = Lethal toxin)
Zinc metallo proteases that inactivates protein kinases
Stimulates Macrophages –TNF alpha and IL – 1 beta – Shock & Death
Death due to oxygen depletion, secondary shock, increased vascular permeability, respiratory failure and cardiac failure.
Sudden and unexpected.
Clinically three forms of Human anthrax occur
A. Cutaneous anthrax-Hide-porters diseaseA. Pulmonary anthrax-Woo-lsorters diseaseB. Intestinal anthrax
Broadly can be classified into
Non Industrial/Agricultural ( Through infected animals):
Cutaneous anthrax Rarely intestinal anthrax
Industrial Anthrax ( Through animal products):
Mostly through animal products( wools, hair, hides, bones) Likely to develop Cutaneous and pulmonary anthrax ( inhalation)
Cutaneous Anthrax
• Mainly in professionals( Veterinarian, butcher)
• Spores infect skin- a characteristic gelatinous edema develops at the
site (Papule-Vesicle-Malignant Pustule- Necrotic ulcer)
• Hide porter’s disease
• IP – 1 to 12 days, 2000 cases annually
• 95-99% of all human anthrax occur as cutaneous anthrax
• Mortality – 20% untreated , 1% treated (edema/septicaemia)
• 80-90% heal spontaneously ( 2-6wks)
• 0-20% progressive disease – develop septicemia
Cutaneous Anthrax
Pulmonary Anthrax
• Wool sorter’s disease
• Most common source of exposure – industrial exposure to spores specially tanneries, wool handlers• Hemorrhagic pneumonia, Haemorrhagic mediastinitis• Progress to septicemia very rapidly• Hemorrhagic meningitis –complication
• Acquired through inhalation of spores ( Bioterrorism -aerosol)
• Present with symptoms of severe respiratory infection( High fever & Chest pain)
• Mortality rate is very high > 95%
• Intentional contamination of mails since 2001
October 2001 letter associated Anthrax outbreak
22 cases
11 Inhalational (5 deaths)
11 Cutaneous (No deaths)
Very different distribution compared to naturally occurring disease
Mortality – historically: >95%
Gastrointestinal Anthrax Rare –primitive communities who eat carcasses of animals
dying of anthrax Nausea, anorexia, vomiting, fever Progresses to severe abdominal pain and bloody emesis and
diarrhea Death 2 to 5 days after onset of symptoms Very difficult to diagnose
LAB DIAGNOSIS
Samples:
-Cutaneous:- vesicular fluid on swabs
- full thickness punch biopsy in 10%
buffered formalin
-Gastrointestinal:- stool, blood, peritoneal fluid, splenic & node aspirate
-Inhalational:- pleural fluid
- blood/paired sera
-Meningeal:- CSF
LAB DIAGNOSIS
Disposable gloves, aprons, boots, face shields, respirators (autoclaved)
• Indisposable –
10% formaldehyde
5% glutaraldehyde
fumigation
• Aerosol tight rotors
• Biosafety cabinet
• Level 2 / 3 practices
MicroscopyStainsGram stain
Carcasses 1 or 2 day old Aspirate blood - MacFadyean stain for bacilli Direct demonstration by IFA
Spore stain
Culture:
SBAGelatin stab culture Polymyxin lysozyme EDTA thallous acetate (PLET agar)
ELISA: based on anthrax toxin ( PA, LF and EF) for routine confirmation of antibodies
Molecular techniques ( Only in the referral laboratories): RFLP PCR Fingerprinting
Animal Inoculation: Guinea pig and mice inoculation
TREATMENT
Antibiotics therapy is effective in human cases but rarely succeeds in animals –not started sufficiently early
Antibiotic treatment is effective in cutaneous anthrax
Penicillin, tetracyclines, erythromycin and fluoroquinolones are effective
Inhalation anthrax can be effectively treated with antibiotics administered prior to lymphatic spread or septicemia
Prophylaxis Hygiene- improvement of factory environment Proper sterilisation of animal products Carcasses- buried deep in quicklime or cremated to prevent soil
contamination
Immunisation: Prevention of anthrax in animals- Original Pasteur’s anthrax vaccine Anthrax bacillus attenuated by growth at 42-43˚C
Spore vaccines: Sterne vaccine-avirulent, mutant strain Mazzucchi vaccine-spores of stable Carbazoo strain in 2%
saponin
DIAGNOSIS(contd….)
Characteristic B. anthracisB. cereus and B.thuringiensis
growth requirement for thiamin + -hemolysis on sheep blood agar - +glutamyl-polypeptide capsule + -lysis by gamma phage + -motility - +growth on chloralhydrate agar - +string-of-pearls test + -